Feijoa fruit extract

ABSTRACT

The invention relates to a feijoa fruit extract. In particular the invention relates to therapeutic uses of a feijoa fruit extract, processes for preparing the extract, and compositions comprising the extract. The feijoa fruit extract of the invention can be used in the treatment and prevention of rheumatoid arthritis and Type-2 diabetes.

RELATED APPLICATIONS

This application is a 371 filing of Patent Cooperation TreatyApplication No. PCT/IB2013/054727, filed Jun. 10, 2013, which claimspriority to New Zealand Patent Application Nos. 600560, filed Jun. 11,2012 and 600561, filed Jun. 11, 2012, the contents of each of which areincorporated herein by reference in their entirety.

1. TECHNICAL FIELD

The invention relates to a feijoa fruit extract. In particular theinvention relates to therapeutic uses of a feijoa fruit extract, aprocess for preparing the extract, and compositions comprising theextract.

2. BACKGROUND

Diabetes mellitus and obesity have become major global health concerns.Approximately 7% of the American population are affected by diabetes,and 600 million people were estimated to be obese in 2010. The number ofpeople affected by these conditions is expected to increasesubstantially over the next 25 years.

Type-2 diabetes is the most common form of diabetes and accounts for 90%of all cases. Type-2 diabetes increases the risk of cardiovascularmorbidity and mortality, due to the detrimental vascular effects ofprolonged exposure to a hyperglycemic environment, as well as the higherprevalence of associated cardiovascular risk factors: atherosclerosis,hypertension, and clotting abnormalities.

Sufferers are often overweight and suffer from hypertension (high bloodpressure) and hyperlipidaemia (elevated or abnormal blood lipid levels).These conditions have a major impact on the well-being and lifestyle ofsufferers. Hyperglycemia sustained over a prolonged period of time cancause severe damage to the eyes, kidneys, nerves, and pose an increasedrisk of further complications such as heart attack, stroke, amputationof lower limbs and death. Risks of hyperlipidaemia from diabetes caninclude pancreatitis, plaque formation, hardening of the arteries and anincreased risk of cardiovascular disease.

Obesity is a key contributor to Type-2 diabetes. At present, currentobesity treatments, except for surgical removal of the tissue, havefailed to result in a sustained reduction of obesity. Furthermore, drugsused for treating obesity may have serious side effects.

Treatment initially involves changes in lifestyle, diet, or theadministration of oral medications. Patients are often treatednon-specifically which results in undesirable side-effects such asdiarrhoea and nausea. The search for new treatments remains ongoing.Natural products and extracts occupy a special place in the market assuch treatments are thought to be less toxic and more acceptable to thegeneral population than pharmaceutical therapies.

Rheumatoid arthritis has also become a major global health concern.Approximately 1% of the American population are affected by rheumatoidarthritis. The number of people affected by this condition is expectedto increase substantially over the next 25 years.

Rheumatoid arthritis is a systemic autoimmune disease characterised bythe chronic inflammation and destruction of joints and surroundingtissue. It is associated with increased mortality. The exact origin ofrheumatoid arthritis onset remains unknown. However, rheumatoidarthritis involves a systemic imbalance between pro- andanti-inflammatory cytokine activities, causing the induction of chronicinflammation and joint damage. Therefore, most therapeutic agents for RAhave been designed to modulate cytokine levels; for example, tumournecrosis factor-α (TNF-α) and interleukin-1 (IL-1) receptor antagonistshave shown promising activity in the prevention of joint destruction byRA. However, humoral immune suppressions by antagonists exhibittherapeutic effects only for a limited period. Treatment for rheumatoidarthritis is often long term, involving both medication and physicaltherapy.

Some research suggests a connection between rheumatoid arthritis andType-2 diabetes. People with rheumatoid arthritis show an increased riskof insulin resistance and Type-2 diabetes (Wasko M. C.; Kay, J.; Hsia,H. C. and Rahman, M. U. Arthritis Care Res (Hoboken) 2011,63(4):512-21).

The link may be due to the inflammation that occurs with rheumatoidarthritis, which may cause insulin resistance. Some drugs used to treatrheumatoid arthritis may also increase the risk of Type-2 diabetes.Steroids such as prednisone can induce Type-2 diabetes in those alreadyat risk of it. The symptoms of rheumatoid arthritis may also contribute.Joint pain and general fatigue discourage physical exercise making itharder for subjects to maintain cardiovascular fitness and manage weightgain.

There is therefore a need for alternative therapies that can helpprevent and manage these conditions. It is an object of the invention toprovide a natural fruit extract that goes at least some way towardsmeeting this need, or at least provides the public with a useful choice.

3. SUMMARY OF INVENTION

The invention generally provides uses of feijoa fruit extract inmedicinal applications associated with the treatment of Type-2 diabetesand related conditions, and the treatment of rheumatoid arthritis andrelated conditions. The feijoa extract of the invention can be used totreat both conditions simultaneously, or can be used to alleviatesymptoms in a subject with rheumatoid arthritis, while simultaneouslyreducing the risk that the subject will develop Type-2 diabetes.

In a first aspect, the invention provides a method of lowering serumlipids in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract. The patient may be a humanpatient with Type-2 diabetes.

In a second aspect, the invention provides a method of lowering serumglucose in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract. The patient may be a humanpatient with Type-2 diabetes.

In a third aspect, the invention provides a method of lowering bloodpressure in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract. The patient may be a humanpatient with Type-2 diabetes.

In a fourth aspect, the invention provides a method of preventing ortreating a disease or disorder in a human patient with Type-2 diabeteswhere it is desirable to lower blood pressure, comprising administeringto the human patient an effective amount of feijoa fruit extract. Thedisease or disorder may be hypertension or any disease or disorderassociated with elevated blood pressure.

In a fifth aspect, the invention provides a method of preventing ortreating a disease or disorder in a human patient with Type-2 diabeteswhere it is desirable to lower blood lipids, for example cholesterol andtriglycerides, comprising administering to the human patient aneffective amount of feijoa fruit extract. The disease or disorder may behyperlipidaemia or any other disease or disorder associated withelevated blood lipids.

In a sixth aspect, the invention provides a method of preventing ortreating a disease or disorder in the human patient with Type-2 diabeteswhere it is desirable to lower blood glucose, comprising administeringto a human patient an effective amount of feijoa fruit extract. Thedisease or disorder may be hyperglycemia or any other disease ordisorder associated with elevated blood glucose levels. The inventionfurther provides a method of reducing HbA1c level in the patient,comprising administering to a patient an effective amount of feijoafruit extract. The patient may be a human patient with Type-2 diabetes.

In an seventh aspect, the invention provides a method of amelioratingthe symptoms of diabetes associated with hyperglycemia, hyperlipidaemiaand/or hypertension in a human patient with Type-2 diabetes, comprisingadministering to the human patient an effective amount of feijoa fruitextract.

In an eighth aspect, the invention provides a method of ameliorating thesymptoms of metabolic syndrome associated with hyperglycemia,hyperlipidaemia and/or hypertension in a human patient, comprisingadministering to the human patient an effective amount of feijoa fruitextract. The patient may be a human patient with Type-2 diabetes.

In a ninth aspect the invention provides a method for preventing orreducing obesity in a patient. The patient may be a human patient.

In a tenth aspect the invention provides a method for preventing ortreating a disease or disorder associated with obesity.

In an eleventh aspect the invention provides a method of reducinghepatic total cholesterol levels in a patient. The patient may be anobese human patient.

In a twelfth aspect, the invention provides a method of ameliorating thesymptoms of hyperglycemia, hyperlipidaemia and/or hypertensionassociated with obesity in a human patient, comprising administering tothe human patient an effective amount of feijoa fruit extract. Thepatient may be a human patient with Type-2 diabetes.

In the above aspects, in one embodiment the patient may be sufferingfrom rheumatoid arthritis.

In a thirteenth aspect, the invention provides a method of regulatingimmune function in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract.

In a fourteenth aspect, the invention provides a method of treating adisease or disorder associated with immunosenescence in a patient,comprising administering to the patient an effective amount of feijoafruit extract.

In a fifteenth aspect, the invention provides a method of preventing ortreating the symptoms of rheumatoid arthritis, comprising administeringto the patient an effective amount of feijoa fruit extract.

In a sixteenth aspect, the invention provides for a method of reducingthe levels of inflammatory cytokines associated with rheumatoidarthritis, comprising administering an effective amount of feijoa fruitextract to a patient. The patient may be a human patient with rheumatoidarthritis.

In a seventeenth aspect, the invention provides a method of reducingsynovial hyperplasia associated with rheumatoid arthritis, comprisingadministering an effective amount of feijoa fruit extract to a patient.The patient may be a human patient with rheumatoid arthritis.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for lowering serum lipids in apatient. The patient may be a human patient with Type-2 diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for lowering serum glucose in apatient. The patient may be a human patient with Type-2 diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for lowering blood pressure in apatient. The patient may be a human patient with Type-2 diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for preventing or treating a diseaseor disorder where it is desirable to lower blood pressure. The diseaseor disorder may be hypertension or any disease or disorder associatedwith elevated blood pressure. The patient may be a human patient withType-2 diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for preventing or treating a diseaseor disorder where it is desirable to lower blood lipids, for examplecholesterol and triglycerides. The disease or disorder may behyperlipidaemia or any other disease or disorder associated withelevated blood lipids. The patient may be a human patient with Type-2diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for preventing or treating a diseaseor disorder where it is desirable to lower blood glucose. The disease ordisorder may be hyperglycemia or any other disease or disorderassociated with elevated blood glucose levels. The invention furtherprovides the use of feijoa fruit extract in the manufacture of amedicament for reducing HbA1c level in a patient. The patient may be ahuman patient with Type-2 diabetes.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for ameliorating the symptoms ofdiabetes associated with hyperglycemia, hyperlipidaemia and/orhypertension in a human patient.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for ameliorating the symptoms ofmetabolic syndrome associated with hyperglycemia, hyperlipidaemia and/orhypertension in a human patient. The patient may be a human patient withType-2 diabetes.

In another aspect the invention the use of feijoa fruit extract in themanufacture of a medicament for preventing or reducing obesity in apatient. The patient may be a human patient.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for preventing or treating a diseaseor disorder associated with obesity.

In still another aspect the invention provides the use of feijoa fruitextract in the manufacture of a medicament for reducing hepatic totalcholesterol levels in a patient. The patient may be an obese humanpatient.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for ameliorating the symptoms ofhyperglycemia, hyperlipidaemia and/or hypertension associated withobesity in a human patient. The patient may be a human patient withType-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in lowering serum lipids in a patient. The patient may be a humanpatient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in lowering serum glucose in a patient. The patient may be a humanpatient with Type-2 diabetes

In still another aspect the invention provides feijoa fruit extract foruse in lowering blood pressure in a patient. The patient may be a humanpatient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in preventing or treating a disease or disorder where it isdesirable to lower blood pressure. The disease or disorder may behypertension or any disease or disorder associated with elevated bloodpressure. The patient may be a human patient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in preventing or treating a disease or disorder where it isdesirable to lower blood lipids, for example cholesterol andtriglycerides. The disease or disorder may be hyperlipidaemia or anyother disease or disorder associated with elevated blood lipids. Thepatient may be a human patient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in preventing or treating a disease or disorder where it isdesirable to lower blood glucose. The disease or disorder may behyperglycemia or any other disease or disorder associated with elevatedblood glucose levels. The invention further provides feijoa fruitextract for use in reducing HbA1c level in a patient. The patient may bea human patient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in ameliorating the symptoms of diabetes associated withhyperglycemia, hyperlipidaemia and/or hypertension in a human patient.

In still another aspect the invention provides feijoa fruit extract foruse in ameliorating the symptoms of metabolic syndrome associated withhyperglycemia, hyperlipidaemia and/or hypertension in a human patient.The patient may be a human patient with Type-2 diabetes.

In still another aspect the invention provides a feijoa fruit extractfor use in preventing or treating obesity.

In still another aspect the invention provides a feijoa fruit extractfor use in preventing or treating a disease or disorder associated withobesity.

In still another aspect the invention provides a feijoa fruit extractfor use in reducing hepatic total cholesterol levels in a patient. Thepatient may be an obese human patient.

In still another aspect the invention provides feijoa fruit extract foruse in ameliorating the symptoms of obesity associated withhyperglycemia, hyperlipidaemia and/or hypertension in a human patient.The patient may be a human patient with Type-2 diabetes.

In still another aspect the invention provides feijoa fruit extract foruse in regulating immune function in a patient.

In still another aspect the invention provides feijoa fruit extract foruse in preventing or treating a disease or disorder associated withimmunosenescence in a patient.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for regulating immune function in apatient.

In another aspect the invention provides the use of feijoa fruit extractin the manufacture of a medicament for preventing or treating thesymptoms of rheumatoid arthritis.

In another aspect, the invention provides the use of feijoa fruitextract for reducing the levels of inflammatory cytokines associatedwith rheumatoid arthritis, comprising administering an effective amountof feijoa fruit extract to a patient. The patient may be a human patientwith rheumatoid arthritis.

In another aspect, the invention provides the use of feijoa fruitextract for reducing synovial hyperplasia associated with rheumatoidarthritis, comprising administering an effective amount of feijoa fruitextract to a patient. The patient may be a human patient with rheumatoidarthritis.

In still another aspect the invention provides feijoa fruit extract foruse in regulating immune function in a patient.

In another aspect the invention provides feijoa fruit extract for use inpreventing or treating rheumatoid arthritis.

In another aspect, the invention provides feijoa fruit extract for usein lowering inflammatory cytokines associated with rheumatoid arthritis,comprising administering an effective amount of feijoa fruit extract toa patient. The patient may be a human patient with rheumatoid arthritis.

In another aspect, the invention provides feijoa fruit extract for usein reducing synovial hyperplasia associated with rheumatoid arthritis,comprising administering an effective amount of feijoa fruit extract toa patient. The patient may be a human patient with rheumatoid arthritis.

In yet another aspect the invention provides a composition for loweringserum lipids in a patient, comprising an effective amount of feijoafruit extract. The patient may be a human patient with Type-2 diabetes.

In yet another aspect the invention provides a composition for loweringserum glucose in a patient, comprising an effective amount of feijoafruit extract. The patient may be a human patient with Type-2 diabetes.

In yet another aspect the invention provides a composition for loweringblood pressure in a patient, comprising an effective amount of feijoafruit extract. The patient may be a human patient with Type-2 diabetes.

In yet another aspect the invention provides a composition forpreventing or treating a disease or disorder where it is desirable tolower blood pressure, comprising an effective amount of feijoa fruitextract. The disease or disorder may be hypertension or any disease ordisorder associated with elevated blood pressure. The patient may be ahuman patient with Type-2 diabetes.

In yet another aspect the invention provides a composition forpreventing or treating a disease or disorder where it is desirable tolower blood lipids, for example cholesterol and triglycerides,comprising an effective amount of feijoa fruit extract. The disease ordisorder may be hyperlipidaemia or any other disease or disorderassociated with elevated blood lipids. The patient may be a humanpatient with Type-2 diabetes.

In yet another aspect the invention provides a composition forpreventing or treating a disease or disorder where it is desirable tolower blood glucose, comprising an effective amount of feijoa fruitextract. The disease or disorder may be hyperglycemia or any otherdisease or disorder associated with elevated blood glucose levels. Theinvention further provides a composition for reducing HbA1c level in apatient. The patient may be a human patient with Type-2 diabetes.

In yet another aspect the invention provides a composition forameliorating the symptoms of diabetes associated with hyperglycemia,hyperlipidaemia and/or hypertension in a human patient, comprising aneffective amount of feijoa fruit extract.

In yet another aspect the invention provides a composition forameliorating the symptoms of metabolic syndrome associated withhyperglycemia, hyperlipidaemia and/or hypertension in a human patient,comprising an effective amount of feijoa fruit extract. The patient maybe a human patient with Type-2 diabetes.

In yet another aspect the invention provides a composition forameliorating the symptoms of hyperglycemia, hyperlipidaemia and/orhypertension associated with obesity in a human patient, comprising aneffective amount of feijoa fruit extract. The patient may be a humanpatient with Type-2 diabetes.

In another aspect the invention provides a composition for preventing ortreating obesity, comprising an effective amount of feijoa fruitextract.

In another aspect the invention provides a composition for preventing ortreating a disease or disorder associated with obesity, comprising aneffective amount of feijoa fruit extract.

In another aspect the invention provides a composition for reducinghepatic total cholesterol in a patient, comprising an effective amountof feijoa fruit extract. The patient may be an obese human patient.

In yet another aspect the invention provides a composition forregulating immune function in a patient, comprising an effective amountof feijoa fruit extract.

In yet another aspect the invention provides a composition forpreventing or treating a disease or disorder associated withimmunosenescence in a patient, comprising an effective amount of feijoafruit extract. In another aspect the invention provides feijoa fruitextract for use in the manufacture of a medicament.

In yet another aspect the invention provides a composition forregulating immune function in a patient, comprising an effective amountof feijoa fruit extract.

In another aspect the invention provides a composition for preventing ortreating rheumatoid arthritis, comprising an effective amount of feijoafruit extract.

In another aspect, the invention provides a composition for reducing thelevels of inflammatory cytokines associated with rheumatoid arthritis,comprising an effective amount of feijoa fruit extract. The patient maybe a human patient with rheumatoid arthritis.

In another aspect, the invention provides a composition for reducingsynovial hyperplasia associated with rheumatoid arthritis, comprising aneffective amount of feijoa fruit extract. The patient may be a humanpatient with rheumatoid arthritis.

In another aspect the invention provides feijoa fruit extract for use inthe manufacture of a medicament.

In another aspect the invention provides the use of feijoa fruit extractas a medicament.

In yet another aspect, the invention provides a composition comprisingfeijoa fruit extract. The composition may be a food or food product.Alternatively, the composition may be a dietary supplement, such as anutraceutical or other nutritional composition.

In yet another aspect, the invention provides a pharmaceuticalcomposition comprising feijoa fruit extract, admixed with one or morepharmaceutically acceptable excipients.

In a further aspect, the invention provides the use of feijoa fruitextract as a nutraceutical, such as a dietary supplement, or as anactive ingredient in the preparation of medical or functional foods andbeverages.

Preferably the feijoa fruit extract used in any of the above-describedmethods, uses and compositions is provided in the form of apharmaceutical composition, a nutraceutical, e.g. a dietary supplement,or a food product, e.g. a medical or functional food or beverage.

In one embodiment, the feijoa fruit extract comprises at least onecompound selected from the group consisting of: catechin, epicatechin,gallocatechin, proanthocyanidins, ellagic acid, pedunculagin, luteolinand related ellagitannins. More preferably the feijoa fruit extractcomprises two or more compounds selected from the group consisting of:catechin, epicatechin, gallocatechin, proanthocyanidins, ellagic acid,pedunculagin, luteolin and related ellagitannins.

In one embodiment, the feijoa fruit extract comprises at least onecompound selected from the group consisting of: catechin, epicatechin,gallocatechin, proanthocyanidins, flavones, flavanols, ellagic acid,pedunculagin, and related ellagitannins. More preferably the feijoafruit extract comprises two or more compounds selected from the groupconsisting of: catechin, epicatechin, gallocatechin, proanthocyanidins,flavones, flavanols, ellagic acid, pedunculagin and relatedellagitannins.

In one embodiment, the feijoa fruit extract comprises (a) at least oneoligomeric proanthocyanidin (b) at least one ellagitannin, and (c)flavone.

In another embodiment, the feijoa fruit extract comprises (a) about 40to about 70 wt % oligomeric proanthocyanidin and (b) about 10 to about20 wt % ellagitannins.

In one embodiment, the feijoa fruit extract comprises pedunculagin.

In one embodiment, the feijoa fruit extract comprise at least about 50,55, 60, 65, 70, 75, 80, 85, 90 or 95 wt % polyphenol compounds.

In one embodiment the feijoa fruit extract is prepared by a processincluding the steps (i) to (vi) defined below.

In another aspect, the invention provides a process for preparing feijoafruit extract, including the steps of:

-   -   i) contacting whole feijoa fruit, and/or feijoa fruit skin,        and/or feijoa fruit pulp and/or residues of feijoa fruit from        feijoa fruit juicing, with an organic solvent, water or an        aqueous/organic solvent to provide an aqueous, organic or        aqueous/organic extract and a solid residue; and    -   ii) separating the aqueous, organic or aqueous/organic extract        from the solid residue to give a crude aqueous, organic or        aqueous/organic feijoa fruit extract.    -   iii) evaporating the crude aqueous, organic or aqueous/organic        feijoa fruit extract from step (ii) to give a substantially        aqueous feijoa fruit extract concentrate and a precipitate; and    -   iv) separating the substantially aqueous feijoa fruit extract        concentrate from the precipitate.    -   v) contacting the substantially aqueous feijoa fruit extract        from step (iv) with a polymeric resin to adsorb at least one        component from the substantially aqueous feijoa fruit extract;    -   vi) eluting at least one component from the resin with an        organic solvent or a mixture of organic solvents to obtain the        feijoa fruit extract.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows HPLC traces of feijoa fruit extract prepared in Example 1and analysed in Example 2. Upper chromatogram is from diode arraydetector, lower chromatogram is total ion current (electrospray,positive mode) from mass spectrometer.

FIG. 2 shows the radiological images of the paws of (A) normal control,(B) arthritic control, (C) methotrexate-treated and (D) feijoa fruitextract-supplemented mice in the collagen-induced arthritis mouse study(Example 6).

FIG. 3 shows histological images after hematoxylin and eosin (H&E)staining of the joint sections of the paws of (A) normal control, (B)arthritic control, (C) feijoa fruit extract-supplemented and (D)methotrexate-treated mice in the collagen-induced arthritis mouse study(Example 6).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Abbreviations

-   ACE Angiotensin-converting enzyme-   BP Blood Pressure-   CM Culture media-   ConA Concanavalin A-   CVD Cardiovascular disease-   ERK Extracellular signal-regulated kinase-   FBS Fetal Bovine Serum-   FITC Fluorescein Isothiocyanate-   HbA1c Glycosylated Hemoglobin A1c-   HCl Hydrochloric acid-   HDL High density lipoprotein-   IL Interleukin-   IFN Interferon-   IgG1 Immunoglobulin G1-   IκBα Nuclear factor of kappa light polypeptide gene enhancer in    B-cells inhibitor, alpha-   kg Kilograms-   LDL Low density lipoprotein-   LPS Lipopolysaccharide-   MDA Malondialdehyde-   mAb Monoclonal antibody-   MI Mitogenesis Index-   nM Nano molar-   NO Nitric Oxide-   PBS Phosphate buffered saline-   PE Phycoerythrin-   PGE Prostaglandin E2-   PHA Phytohemagglutinin-   RPMI 1640 A commercial liquid medium

5.2 Definitions

The term “feijoa fruit extract” as used herein means an extract of thefeijoa fruit, including the whole fruit, and/or skin of the feijoa fruitand/or feijoa fruit pulp and/or residues of feijoa fruit remaining afterjuicing of the fruit (feijoa fruit pomace). The fruit or parts thereofmay be fresh, frozen (and then thawed) or dried. Juice that is recoveredfrom any processing of the fruit any also be processed to recover afeijoa fruit extract.

The term “feijoa fruit skin” as used herein means the outer waxy coverof the fruit.

The term “feijoa fruit pulp” as used herein means the fleshy contents ofthe feijoa fruit.

The term “feijoa fruit pomace” as used herein refers to the fruitresidue obtained after juicing of the fruit.

The term “patient” includes human and non-human animals.

The terms “treatment”, “treating” and the like include the reduction oralleviation of one or more symptom associated with the disease ordisorder. For example, for hyperglycemia, hyperlipidaemia orhypertension this can mean reduction in serum glucose levels, serumlipid levels or blood pressure, respectively.

The terms “preventing”, “prevention” and the like include the preventionof one or more symptom associated with the disease or disorder.

The term “comprising” as used in this specification and claims means“consisting at least in part of”. When interpreting statements in thisspecification and claims which include the term “comprising”, otherfeatures besides the features prefaced by this term in each statementcan also be present. Related terms such as “comprise” and “comprised”are to be interpreted in similar manner.

6. DETAILED DESCRIPTION

Research on human nutrition has led to an awareness of the healthbenefits of dietary supplements. It is recognised that dietarysupplements containing complex arrays of naturally occurring bioactivecompounds may confer significant long-term health benefits.

Feijoa sellowiana Berg syn. Acca sellowiana, commonly known aspineapple-guava, is a fruit-bearing evergreen shrub relative of tropicalguava. The feijoa plant is indigenous to South America but can be foundin tropical and subtropical dry areas, particularly in New Zealand. Itis grown for its fruit, primarily for the production of juice with asweet and acidic flavour.

The species has assumed some medicinal relevance due to its content ofbiologically and nutritionally interesting compounds. A recent reviewhas shown there are no known reports of the effects of feijoaconsumption on heath, disease prevention or therapy, including diabetesin humans or animals. (Arg{umlaut over (ν)}elles, M. C., Watson, R. R.,Feijoa (pineapple-guava) fruit: A role in health promotion? In: R. R.Watson, V. Preedy, editors, Bioactive Foods and Extracts: CancerTreatment and Prevention, CRC Press, published December 2010, ISBW1439816190)). However, some studies have shown a high correlationbetween the total phenol content and the radical scavenging activity ofextracts of feijoa on cultured cells (Yuka, I., Yumiko, K., Miyo, N.,Takashi K., J. Home. Econ. Japan, 2003, 54, 945-949). Therefore, thefeijoa fruit has joined a growing list of edible plant that are beinginvestigated for their medicinal properties.

The applicants have discovered that feijoa fruit extract has efficacy inthe treatment and prevention of both diabetes and rheumatoid arthritis.

6.1 The Feijoa Fruit Extract

The feijoa fruit extract of the invention is prepared by extracting thefeijoa fruit, including the whole fruit, and/or skin of the feijoa fruitand/or feijoa fruit pulp and/or residues of feijoa fruit remaining afterjuicing of the fruit.

The extraction process may be prepared using standard extractiontechniques known in the art. In one embodiment the feijoa fruit extractis prepared by a process including the steps (i) to (vi) defined below.

In one aspect the invention provides a process for preparing feijoafruit extract, including the steps of:

-   -   i) contacting feijoa fruit, and/or feijoa fruit skin, and/or        feijoa fruit pulp and/or residues of feijoa fruit from feijoa        fruit juicing, with a hydrophilic organic solvent, water or an        aqueous/organic solvent to provide an aqueous, organic or        aqueous/hydrophilic organic extract and a solid residue; and    -   ii) separating the aqueous, organic or aqueous/organic extract        from the solid residue to give a crude aqueous, organic or        aqueous/organic feijoa fruit extract;    -   iii) evaporating the crude aqueous, organic or aqueous/organic        feijoa fruit extract from step (ii) to give a substantially        aqueous feijoa fruit extract concentrate and a precipitate;    -   iv) separating the substantially aqueous feijoa fruit extract        concentrate from the precipitate; and    -   v) fractionating the substantially aqueous feijoa fruit extract        concentrate from step iv) to concentrate at least one component        thereof.

In one embodiment the substantially aqueous feijoa fruit extractconcentrate from step iv) is fractionated using polymeric resinabsorption, chromatographic separation, solvent partitioning orselective precipitation.

In another embodiment the substantially aqueous feijoa fruit extractconcentrate from step iv) may be dried and dissolved in a mixture of anorganic solvent and water, for example 70:30 ethanol:water. The sugarsand other polar material are not soluble in this solvent. Drying theorganic solvent and water mixture will provide a feijoa fruit extractmore concentrated in polyphenol compounds. This procedure can berepeated several times.

In another embodiment the substantially aqueous feijoa fruit extractconcentrate from step iv) may be dissolved in a non-water misciblesolvent, such as ethyl acetate or 1-butanol to yield a non-aqueousfeijoa fruit extract with high polyphenol levels.

In another aspect, the invention provides a process for preparing feijoafruit extract, including the steps of:

-   -   i) contacting whole feijoa fruit, and/or feijoa fruit skin,        and/or feijoa fruit pulp and/or residues of feijoa fruit from        feijoa fruit juicing, with an organic solvent, water or an        aqueous/organic solvent to provide an aqueous, organic or        aqueous/organic extract and a solid residue; and    -   ii) separating the aqueous, organic or aqueous/organic extract        from the solid residue to give a crude aqueous, organic or        aqueous/organic feijoa fruit extract;    -   iii) evaporating the crude aqueous, organic or aqueous/organic        feijoa fruit extract from step (ii) to give a substantially        aqueous feijoa fruit extract concentrate and a precipitate;    -   iv) separating the substantially aqueous feijoa fruit extract        concentrate from the precipitate;    -   v) contacting the substantially aqueous feijoa fruit extract        from step (iv) with a polymeric resin to adsorb at least one        component from the substantially aqueous feijoa fruit extract;    -   vi) eluting at least one component from the resin with an        organic solvent or a mixture of organic solvents to obtain the        feijoa fruit extract.

The feijoa fruit extract produced by the process of the invention can bevacuum dried, spray dried or freeze-dried to produce a free-flowingpowder.

In one embodiment, the feijoa fruit extract is a free-flowing powder.

The whole feijoa fruit, and/or feijoa fruit skin and/or feijoa fruitpulp and/or residues of feijoa fruit left over after juicing of thefruit may optionally be minced, mashed, crushed, blended, sliced orchopped prior to step (i). The fruit or fruit portions may be frozen andthawed prior to step (i). They may also be dried prior to step (i).

Feijoa fruit may be juiced (the juice removed from the fruit bysqueezing or other means) prior to step (i), thereby providing saidresidues of feijoa fruit left over after juicing of the fruit, which canbe used in step (i). Juicing can be carried out using a centrifuge,screw press or mechanical press, or other method known in the art. Thede-juicing operation may be combined with an enzyme pre-treatment toassist in the release of polyphenol compounds from the fruit or partsthereof.

In one embodiment the organic solvent is acetone or an alcohol such asmethanol, ethanol, isopropyl alcohol or 1-propanol. Where anaqueous/organic solvent is used, the organic solvent and water can beused in any suitable proportions, such as about 20-90% water.

In one embodiment, the substantially aqueous feijoa fruit extractconcentrate from step iv) is fractionated by contacting it with apolymeric resin. The resin absorbs at least one component from theextract concentrate. The component(s) can then be eluted from the resinwith an organic solvent or mixture of organic solvents to give thefeijoa fruit extract of the invention.

The polymeric resin may optionally be washed with distilled water priorto eluting the at least one component. In one embodiment, the polymericresin is a non-ionic polymeric absorbent resin. Preferably the polymericabsorbent resin is a non-iconic styrene-divinyl benzene co-polymer resinsuch as Mitsubishi HP-20, Mitsubishi Sepabeads SP-70 or Amberlite.Example 1 provides an example of a method of preparing feijoa fruitextract of the invention.

Feijoa is known to contain high levels of polyphenols. Polyphenols are awidely ranging group of biological molecules that play a protective rolein plants. Generally characterised by the presence of multiple and oftenpolyhydroxylated phenol units, there are many different classes ofpolyphenols, with a huge variety of compounds falling within each class.

Each plant species will have a different and characteristic polyphenolprofile which will also vary to an extent depending on the environmentalconditions it is grown under. Different processing methods may alsoprovide plant material with different polyphenol profiles. Therefore, itis not feasible to identify all of the polyphenol compounds in anextract of a polyphenol-rich plant. However, chromatographic separationof the fruit extract of the invention leads to the isolation andidentification of catechin (III), epicatechin (IV), gallocatechin (V),procyanidin B2 (VI), proanthocyanidin oligomers (VII), ellagic acid, α-and β-pedunculagin as well as other related ellagitannins (see compounds(I) to (VII), below) as characteristic compounds. The components offeijoa fruit extract prepared in Example 1 are isolated and identifiedin Example 2—see Table 1 and FIG. 1 which relate to HPLC traces offeijoa fruit extract.

In one aspect, the feijoa fruit extract comprises at least one compoundselected from the group consisting of: catechin, epicatechin,gallocatechin, proanthocyanidins, flavone, flavanols, ellagic acid,pedunculagin, and related ellagitannins. More preferably the feijoafruit extract comprises two or more compounds selected from the groupconsisting of: catechin, epicatechin, gallocatechin, proanthocyanidins,flavone, flavanols, ellagic acid, pedunculagin and relatedellagitannins.

In particular, the feijoa fruit extract of the invention has been shownto contain large proportions of two large polyphenols—oligomericproanthocyanidins (proanthocyanidin oligomers) and ellagitannins. Thesetwo polyphenol classes act together in the extracts of the invention toprovide surprising efficacy in the treatment and prevention Type-2diabetes and rheumatoid arthritis.

In one aspect, the invention provides a feijoa fruit extract comprising

(a) about 40 to about 70 wt %, (preferably about 50 to about 60 wt %)oligomeric proanthocyanidin and

(b) about 10 to about 20 wt % ellagitannins, on a dry weight basis.

In other words, when the extract is substantially free of water, about40 to about 70 wt % of the extract consists of oligomericproanthocyanidin.

In another aspect, the invention provides a feijoa fruit extractcomprising (a) about 40 to about 70 wt %, (preferably about 50 to about60 wt %) oligomeric proanthocyanidin and (b) about 10 to about 20 wt %ellagitannins, on a total polyphenol basis.

In other words, oligomeric proanthocyanidins make up about 40 to about70 wt % of the total polyphenols present in the extract.

Preferably, the feijoa fruit extract comprises pedunculagin and/orflavone.

In one embodiment, the feijoa fruit extract comprises at least about 50,55, 60, 65, 70, 75, 80, 85, 90, or 95 wt % polyphenol compounds, on adry weight basis. Preferably, the feijoa fruit extract comprises about60 to about 90% polyphenol compounds, on a dry weight basis.

In one embodiment, the feijoa fruit extract comprises (a) about 40 toabout 70 wt % (preferably about 50 to 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins,wherein the feijoa fruit extract comprises about 60 to about 90 wt %(preferably about 70 to about 90 wt %) polyphenol compounds, on a dryweight basis.

In one embodiment, the feijoa fruit extract comprises (a) about 40 toabout 70 wt % (preferably about 50 to 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins,wherein the feijoa fruit extract comprises about 60 to about 90 wt %(preferably about 70 to about 90 wt %) polyphenol compounds.

Advantageously, the feijoa fruit extract of the invention can beprovided to a patient in a variety of forms. For example, the feijoafruit extract can be provided as a nutraceutical, e.g. a dietarysupplement. Alternatively, the feijoa fruit extract can be provided as afood product, e.g. as part of a functional food or beverage. The feijoafruit extract of the invention can be provided as an adjunct toconventional treatments. As a natural food ingredient, it is unlikely toproduce undesirable side-effects such as interfering with the efficacyof conventional pharmaceutical treatments.

Alternatively, the feijoa fruit extract can be provided as apharmaceutical composition. The extract may be formulated into solid orliquid preparations such as tablets, capsules, suppositories, powders,solutions, suspensions and dispersions. In some embodiments the feijoafruit extract is formulated for oral administration as solid or liquidpreparations, for example tablets, capsules, powders, solutions,suspensions or dispersions. Such preparations are well known in the artas are other oral dosage regimes not listed here. In the tablet form thefeijoa fruit extract may be tableted with conventional tablet bases suchas lactose, sucrose and corn starch, together with a binder, adisintegration agent and a lubricant. The binder may be, for example,corn starch or gelatin, the disintegrating agent may be potato starch oralginic acid, and the lubricant may be magnesium stearate. For oraladministration in the form of capsules, diluents such as lactose anddried cornstarch may be employed. Other components such as colourings,sweeteners or flavourings may be added.

When aqueous suspensions are required for oral use, the feijoa fruitextract may be combined with carriers such as water and ethanol, andemulsifying agents, suspending agents and/or surfactants may be used.Colourings, sweeteners or flavourings may also be added.

The compounds may further be administered by means of sustained releasesystems. For example, they may be incorporated into a slowly dissolvingtablet or capsule.

Liquid forms include carriers such as water and ethanol, with or withoutother agents such as pharmaceutically acceptable surfactants orsuspending agents.

In one aspect the invention provides a dosage unit comprising about 150mg feijoa fruit extract.

In another aspect, the invention comprises a dosage unit consistingessentially of 150 mg feijoa fruit extract.

Preferably, the dosage unit is a tablet.

6.2 Use of Feijoa Fruit Exact in Regulating Immune Function

Ageing is clearly associated with a decline in immune function and is amajor contributing factor to high rates of morbidity and mortality inthe elderly. The feijoa fruit extract of the present invention is usefulfor regulating immune function and/or for preventing or treatingdiseases disorders associated with immunosenescence in a patient.

The effect of the feijoa fruit extract of the present invention on theimmune function of mice was evaluated in Example 3. Aged mice fed dietssupplemented with feijoa fruit extract were evaluated for changes inimmune function by assessing production of cytokines, mitogenesis ofsplenocytes and vitamin E levels. IL-2, IL-4, IL-6 and IFN areinflammatory cytokines important in the B-cell production of antibodiesand are key components of the adaptive immune system. Aging is a majorrisk factor for the development of diabetes and its complications andthus reduced inflammatory cytokines also help lower the risk fordiabetes. Example 3 demonstrates that the feijoa fruit extractsupplemented at an appropriate dose causes significant increase insplenic T-cell immune function in aged mice. The feijoa fruit extractalso increases levels of hepatic vitamin E and lowers TNF-α levels. Lowgrade chronic inflammation is a common event in the aged and thepro-inflammatory cytokine modulation observed suggest that feijoa fruitextract helps to treat chronic inflammation as measured by cytokinechanges. Inflammation, typically stimulated by dysregulation in aging ordisease with elevated T-helper 2 cytokines, contributes to arthritis anddiabetes. Immune dysregulation in aged systems can exacerbateinflammation and thus could accentuate rheumatoid arthritis.

In one aspect, the invention provides a method of regulating immunefunction in a patient, comprising administering to the patent aneffective amount of feijoa fruit extract, wherein the feijoa fruitextract comprises (a) about 40 to about 70 wt %, (preferably about 50 toabout 60 wt %) oligomeric proanthocyanidin and (b) about 10 to about 20wt % ellagitannins, on a dry weight basis.

In another aspect, the invention provides a method of treating a diseaseor disorder associated with immunosenescence in a patient, comprisingadministering to the patient an effective amount of feijoa fruitextract, wherein the feijoa fruit extract comprises (a) about 40 toabout 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on adry weight basis.

In another aspect, the invention provides a method of reducing thelevels of inflammatory cytokines in a patient, comprising administeringto the patient an effective amount of feijoa fruit extract, wherein thefeijoa fruit extract comprises (a) about 40 to about 70 wt %,(preferably about 50 to about 60 wt %) oligomeric proanthocyanidin and(b) about 10 to about 20 wt % ellagitannins, on a dry weight basis.

In one aspect, the invention provides a method of regulating immunefunction in a patient, comprising administering to the patent aneffective amount of feijoa fruit extract, wherein the feijoa fruitextract comprises (a) about 40 to about 70 wt %, (preferably about 50 toabout 60 wt %) oligomeric proanthocyanidin and (b) about 10 to about 20wt % ellagitannins, on a total polyphenol basis.

In another aspect, the invention provides a method of treating a diseaseor disorder associated with immunosenescence in a patient, comprisingadministering to the patient an effective amount of feijoa fruitextract, wherein the feijoa fruit extract comprises (a) about 40 toabout 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on atotal polyphenol basis.

In another aspect, the invention provides a method of reducing thelevels of inflammatory cytokines in a patient, comprising administeringto the patient an effective amount of feijoa fruit extract, wherein thefeijoa fruit extract comprises (a) about 40 to about 70 wt %,(preferably about 50 to about 60 wt %) oligomeric proanthocyanidin and(b) about 10 to about 20 wt % ellagitannins, on a total polyphenolbasis.

In the above aspects:

In one embodiment, the feijoa fruit extract comprises about 60 to about90% polyphenol compounds, on a dry weight basis.

In one embodiment, the feijoa fruit extract comprises about 60 to about90 wt % polyphenol compounds.

In one embodiment, the patient is administered 150 mg of feijoa fruitextract daily.

In one aspect, the invention provides a dosage unit for regulatingimmune function in a patient, wherein the dosage unit comprises about150 mg feijoa fruit extract.

In one embodiment, the dosage unit is a tablet. In one embodiment, thedosage unit is to be administered once daily.

6.3 Use of Feijoa Fruit Extract in the Treatment and/or Prevention ofObesity, Type-2 Diabetes and Associated Diseases

The feijoa fruit extract of the present invention has been shown to beuseful in the treatment and/or prevention of diseases and disorders suchas those associated with obesity, metabolic syndrome and Type-2diabetes. Diseases and disorders include those associated with elevatedblood pressure, elevated serum glucose levels and/or elevated serumlipid levels, e.g. hyperglycemia, hyperlipidaemia and hypertension.

The applicants have surprisingly shown that supplementation of the dietof Type-2 diabetes patients with feijoa fruit extract reduces thepatient's blood pressure and results in improved diabetes control.Plasma fasting glucose and HbA1c levels improve and reach statisticalsignificance when compared to a placebo (see Example 4).

Without wishing to be bound by theory, it is thought that theglucose-lowering effect of an extract of feijoa might be mediatedthrough the inhibition of α-glucosidase, thus diminishing glucoseintestinal resorption. Furthermore, feijoa fruit extract of the presentinvention is useful for preventing or reducing obesity through thesuppression of weight gain and lowering of the food efficiency ratio.

The effect of the feijoa fruit extract of the present invention onobesity and related lipid driven risk factors in mice was also evaluated(see Example 5). Those mice receiving the feijoa fruit extract showedlower body weight gain in comparison to the control group.

In one aspect, the invention provides a method for preventing ortreating Type-2 diabetes in a patient, comprising administering to thepatient an effective amount of feijoa fruit extract. In one embodiment,the patient suffers from rheumatoid arthritis.

In another aspect, the invention provides a method for lowering serumlipids in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the feijoa fruitextract comprises (a) about 40 to about 70 wt % (preferably about 50 toabout 60 wt %) oligomeric proanthocyanidin and (b) about 10 to about 20wt % ellagitannins, on a dry weight basis.

In another aspect, the invention provides a method for lowering serumglucose in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the (a) about 40 toabout 70 wt % (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on adry weight basis

In another aspect, the invention provides a method for lowering bloodpressure in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the (a) about 40 toabout 70 wt % (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on adry weight basis.

In another aspect, the invention provides a method for treating orpreventing metabolic syndrome in a patient, comprising administering tothe patient an effective amount of feijoa fruit extract, wherein thefeijoa fruit extract comprises (a) about 40 to about 70 wt % (preferablyabout 50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10to about 20 wt % ellagitannins, on a dry weight basis.

In another aspect, the invention provides a method for lowering serumlipids in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the feijoa fruitextract comprises (a) about 40 to about 70 wt % (preferably about 50 toabout 60 wt %) oligomeric proanthocyanidin and (b) about 10 to about 20wt % ellagitannins, on a total polyphenol basis.

In another aspect, the invention provides a method for lowering serumglucose in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the (a) about 40 toabout 70 wt % (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on atotal polyphenol basis.

In another aspect, the invention provides a method for lowering bloodpressure in a patient, comprising administering to the patient aneffective amount of feijoa fruit extract, wherein the (a) about 40 toabout 70 wt % (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on atotal polyphenol basis.

In another aspect, the invention provides a method for treating orpreventing metabolic syndrome in a patient, comprising administering tothe patient an effective amount of feijoa fruit extract, wherein thefeijoa fruit extract comprises (a) about 40 to about 70 wt % (preferablyabout 50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10to about 20 wt % ellagitannins, on a total polyphenol basis.

In one embodiment of the above aspects, the patient is a human withType-2 diabetes.

In another aspect the invention provides a method of treating orpreventing Type-2 diabetes in a patient, comprising administering to thepatient an effective amount of feijoa fruit extract, wherein the feijoafruit extract comprises (a) about 40 to about 70 wt % (preferably about50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10 toabout 20 wt % ellagitannins, on a dry weight basis.

In another aspect the invention provides a method of treating orpreventing Type-2 diabetes in a patient, comprising administering to thepatient an effective amount of feijoa fruit extract, wherein the feijoafruit extract comprises (a) about 40 to about 70 wt % (preferably about50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10 toabout 20 wt % ellagitannins, on a total polyphenol basis.

In the above aspects:

In one embodiment, the feijoa fruit extract comprises about 60 to about90% polyphenol compounds, on a dry weight basis.

In one embodiment, the feijoa fruit extract comprises about 60 to about90 wt % polyphenol compounds.

In one embodiment, the patient is administered 150 mg of feijoa fruitextract daily.

In one aspect, the invention produces a dosage unit for treating orpreventing Type-2 diabetes in a patient, wherein the dosage unitcomprises about 150 mg feijoa fruit extract.

In one embodiment, the dosage unit is a tablet. In one embodiment, thedosage unit is to be administered once daily.

6.4 Use of Feijoa Fruit Extract in the Treatment and/or Prevention ofRheumatoid Arthritis

The feijoa fruit extract of the present invention is also useful in thetreatment and/or prevention of diseases and disorders such as thoseassociated with rheumatoid arthritis.

The effect of the feijoa fruit extract of the present invention oninduced rheumatoid arthritis in mice is evaluated in Example 6. Thecollagen-induced arthritis (CIA) model has been used extensively toelucidate the pathogenic mechanisms that are relevant to humanrheumatoid arthritis, and is widely used for the evaluation of potentialanti-rheumatic agents. The acute stage of CIA is characterised byincreased levels of mRNA for pro-inflammatory cytokines in the joints,such as TNF-α, IL-1β, and IFN-γ. Many of the major pro-inflammatorycytokines that are produced in the rheumatoid synovium, are linked to anetwork or cascade with TNF-α at its apex. IL-1β was previously shown tobe important in cartilage and bone destruction.

Histological studies show that feijoa fruit extract protects againstsynovial hyperplasia—an increase in destructive cytokine production insynovial fluid that can facilitate the onset of rheumatoid arthritis.Radiologic studies show that feijoa fruit extra protects against jointdeformity and soft tissue swelling in CIA mice. Feijoa fruit extractsupplementation also decreases IL-2 and IFN-γ levels compared to thearthritic control group (see Table 9).

In one aspect the invention provides a method of preventing or treatingthe symptoms of rheumatoid arthritis, comprising administering to thepatient and effective amount of feijoa fruit extract, wherein the feijoafruit extract comprises (a) about 40 to about 70 wt %, (preferably about50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10 toabout 20 wt % ellagitannins, on a dry weight basis.

In another aspect of the invention provides a method for reducing thelevels of inflammatory cytokines associated with rheumatoid arthritis,comprising administering to the patient and effective amount of feijoafruit extract, wherein the feijoa fruit extract comprises (a) about 40to about 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on adry weight basis.

In another aspect of the invention provides a method of reducingsynovial hyperplasia associated with rheumatoid arthritis, comprisingadministering to the patient an effective amount of feijoa fruitextract, wherein the feijoa fruit extract comprises (a) about 40 toabout 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on adry weight basis.

In one aspect the invention provides a method of preventing or treatingthe symptoms of rheumatoid arthritis, comprising administering to thepatient and effective amount of feijoa fruit extract, wherein the feijoafruit extract comprises (a) about 40 to about 70 wt %, (preferably about50 to about 60 wt %) oligomeric proanthocyanidin and (b) about 10 toabout 20 wt % ellagitannins, on a total polyphenol basis.

In another aspect of the invention provides a method for reducing thelevels of inflammatory cytokines associated with rheumatoid arthritis,comprising administering to the patient and effective amount of feijoafruit extract, wherein the feijoa fruit extract comprises (a) about 40to about 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on atotal polyphenol basis.

In another aspect of the invention provides a method of reducingsynovial hyperplasia associated with rheumatoid arthritis, comprisingadministering to the patient an effective amount of feijoa fruitextract, wherein the feijoa fruit extract comprises (a) about 40 toabout 70 wt %, (preferably about 50 to about 60 wt %) oligomericproanthocyanidin and (b) about 10 to about 20 wt % ellagitannins, on atotal polyphenol basis.

In the above aspects:

In one embodiment, the feijoa fruit extract comprises about 60 to about90% polyphenol compounds, on a dry weight basis.

In one embodiment, the feijoa fruit extract comprises about 60 to about90 wt % polyphenol compounds.

In one embodiment, the patient is administered 150 mg of feijoa fruitextract daily.

In one aspect, the invention provides a dosage unit for preventing ortreating rheumatoid arthritis, wherein the dosage unit comprises about150 mg feijoa fruit extract.

In one embodiment, the dosage unit is a tablet. In one embodiment, thedosage unit is to be administered daily.

The feijoa fruit extract may be administered to a patient by a varietyof routes, including orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally or via an implanted reservoir.Preferably, the feijoa fruit extract is administered orally, in tabletform. The amount of feijoa fruit extract to be administered will varywidely according to the nature of the patient and the nature and extentof the disorder to be treated. Typically the daily dosage of feijoafruit extract for an adult human will be in the range of less than about1 to about 2000 milligrams, preferably about 0.1 to about 2000milligrams, more preferably about 0.1 to about 1000 milligrams, morepreferably about 50 to about 500 milligrams, more preferably about 100to about 500 milligrams, more preferably about 150 to about 250milligrams, most preferably about 150 milligrams. The specific dosagerequired for any particular patient will depend upon a variety offactors, including the patient's age, body weight, general health, sex,etc.

The invention is further described with reference to the followingexamples. However, it is to be appreciated that the invention is notlimited to these examples.

7. EXAMPLES Example 1: Preparation of Feijoa Fruit Extract

The extract of feijoa was prepared and isolated from fresh feijoa fruit.Whole feijoa fruit, skin, flesh, fruit residues left from juicingoperations or various mixtures of these were minced in a blender. To theresulting pulp was added approximately an equal volume of water,alcohol, a water miscible organic solvent such as acetone or an aqueousmixture of alcohol or organic solvent. In this example, food gradeethanol was used. The mixture was stirred occasionally during the firsthour and left to soak overnight. The mixture was filtered and thefiltrate concentrated at 40° C. under vacuum. The concentrate wasfiltered to remove any precipitated material and then passed through acolumn of polymeric resin, Mitsubish, HP20, in this example. Distilledwater was passed through the column to remove sugars and other polarmaterials. The absorbed compounds were eluted from the column withethanol and concentrated under reduced pressure to give a darkconcentrate. The concentrate was freeze-dried to give the feijoa fruitextract as a light brown powder. The extract was characterised by HPLCusing diode array detection (FIG. 1 and Table 1) and total ion currentdetection (TOF-MS ES+). A Kinetex 2.6μ. C18 (50×3.0 mm) column is usedwith a flow rate of 0.3 mL/min at 40° C.

Example 2: Determination of Components of Feijoa Fruit Extract

The components of the feijoa fruit extract were isolated by repeatedpreparative column chromatography using size exclusion chromatography onSephadex LH 20 and Mitsubishi HP20. Their identification was determinedby the use of Nuclear magnetic resonance spectroscopy and wherenecessary the chemical structures of the elucidated components wereconfirmed by mass spectrometry. A liquid Chromatography-MassSpectrometry chemical composition profile of the feijoa fruit extractwas made using Kinetex 2.6μ C18 (50×3.0 mm) column at 40° C. with a flowrate of 0.3 mL/min. The solvent mixture consisted of solvent A (H₂O+0.1%formic acid) and solvent B (acetonitrile+0.1% formic acid) with theprogram 0-30 min, 0-100%. Detection was made by diode array and totalion current (TIC). Chromatographic separation of feijoa fruit extractled to the isolation and identification of catechin, epicatechin,gallocatechin, procyanidin B2, procyanidin oligomers, ellagic acid, α-and β-pedunculagin as well as other related ellagitannins (FIG. 1 andTable 1).

TABLE 1 HPLC of feijoa extract Retention time (mins) Compound 4.40α-pedunculagin 5.30 β-pedunculagin 5.77 catechin 6.50 gallocatechin 6.80catechin B2 6.81 epicatechin 8.30, 8.77, 9.92 ellagitannins 5.0-9.0procyanidin oligomers 15.3  flavone

The various components were identified using the techniques attiredbelow.

Ellagic Acid Determination

This was performed by acid hydrolysis of the feijoa fruit extract orconcentrate and subsequent analysis of the resulting ellagic acid byHPLC. 80 uL of extract was transferred to a 15 mL glass centrifuge tube,and 250 uL of 2.6 N HCl was added and the tube sealed with a screw cap.The sample was hydrolysed by heating in a water bath at 90 C for 2hours. The tubes were removed and cooled immediately and stored at 4 Cfor 1 hour. The hydrolysates were then extracted 2× with ethyl acetate(1 mL). The combined ethyl acetate extracts were dried under nitrogenand taken up in 1 mL methanol which were analysed by HPLC. The majorpeak in the 360 nm chromatogram (Waters UPLC, Kinetex column,acetonitrile: water gradient each with 0.1% formic acid) was ellagicacid. The levels of ellagic acid were compared with the original extractmade up at 4 mg/mL.

HPLC Determination of Pedunculagin, General Ellagic Acid Compounds andFlavone

Pedunculagin exists as two interconverting forms and is generally seenas two major peaks in the early part of the chromatogram. The peaksharpness is enhanced when the samples are prepared in water or 90:10water:ethanol. The relative pedunculagin content is estimated from theintegration of these two peaks in the 280 nm chromatogram.

The general ellagic acid compounds are seen as a set of peaks eluting inthe middle of the chromatogram and having absorbance maxima at 360 nm.The relative ellagic acid compound content is estimated from theintegration of this set of peaks in the 360 nm chromatogram.

Flavone is a single peak late in the chromatogram with a characteristicuv spectrum. The relative flavone content is estimated from theintegration of this peak in the 280 nm chromatogram.

HPLC Determination of Proanthocyanidin Oligomers (PACO)

This complex set of compounds appear in most chromatograms as a humpacross much of the chromatogram. However if a special gradient is usedthe PACO appear as an isolated hump late in the chromatogram. Theestimation of PACO content from the peak area under this hump isconsistent with measurements made using other techniques. The relativePACO content is estimated from the integration of this hump in the 280nm chromatogram and comparison.

Colorimetric Determination of Proanthocyanidin Oligomers (PACO)

This determination was performed using the procedure described in PriorR L, Fan E, Ji H, Howell A, Nio C, Payne M J, Reed J. Multi-laboratoryvalidation of a standard method for quantifying proanthocyanidins incranberry powders. J Sci Food Agric. 2010 90(9):1473-8). The sample wassallowed to react with 4-dimethylaminocinnamaldehyde (DMAC) in acidicethanol and the coloured product detected by absorption spectroscopy.Samples of the test solutions (70 uL) were placed in one column of thewells of a 96-well microplate and were then diluted across the plate bythe addition and mixing of an equal volume of 70:30 ethanol:water. TheDMAC reagent (210 uL of 50 mg in 50 mL acidified ethanol solution) wasadded to each well and the plate immediately placed in the plate reader.The plate reader wass set up to shake the plate and read the absorbanceat 640 nm every minute for 30 minutes. The absorbance reading generallyreaches a maximum at about 20 minutes. The final readings at 30 minuteswere used for the calculations. The dilution curves allowed comparisonsto be made for the most appropriate concentration and absorbance(0.2-1.2 A.U.).

Example 3: Immune Function in Mice

The effect of the feijoa fruit extract on the immune function in agedmice was evaluated. Aged mice at 48 weeks old, were kept at 20° C. to22° C. at 50% humidity during the experiment. Mice were divided into 2groups and fed the following diets: basic diet or basic dietsupplemented with the feijoa fruit extract at 1.2 mg/day/mouse. Thefeijoa fruit extract was prepared in accordance with Example 1. A thirdgroup of young mice were also included for comparison (basic diet only).The amount of food intake and body weight is recorded once a week.

After 32 weeks of feeding, all mice were sacrificed under anaesthesiawith Nembutal (0.1 mg/100 g body weight). The spleen is isolated andweighed (Table 2).

TABLE 2 Mice Group Mouse Body Weight Age Treatment (g) Spleen (mg) Liver(g) Heart (mg) Young Chow diet 26.36 ± 3.20  89.71 ± 16.25 1.46 ± 0.23153.48 ± 14.12 Aged Chow diet 43.08 ± 2.30 174.95 ± 15.15 1.76 ± 0.15163.88 ± 16.81 Aged Feijoa fruit 44.2 ± 3.1 133.5 ± 19.2 1.7 ± 0.2 160.2± 12.9 extract Mice supplemented with Feijoa fruit extract consumed 6 gmfood/day, which equates to 1.8 mg of extract/day. The feijoa fruitextract was added to the diet at a dose of 300 mg/kg. Data indicatesmean ± SD from 6 mice per group. *Shows the statistical significancecompared to aged control determined by unpaired Students t-test. Therewere no significant differences in the volume of water and diet consumedduring the trial.ELISA Assay for Cytokines

The production of IL-2, IL-4, IL-6, and IFN-γ from mitogen-stimulatedsplenocytes was determined as previously described (Chouaib, S.; Welte,K.; Mertelsman, R.; Dupont, B.; J. Immunol., 1985, 135, 1172-1179).Spleens were gently teased with forceps in the culture medium producinga suspension of spleen cells (CM, RPMI 1640 containing 10% fetal bovineserum, 2 mM L-glutamine, 1×10⁵ units/L of penicillin and streptomycin).The red blood cells were lysed with 3 mL of lysing buffer (0.16 mol/Lammonia chloride Tris buffer, pH 7.2) at 37° C. for 5 minutes. The cellswere then washed with CM twice. The cell concentrations were counted andadjusted to 2×10⁶ cells/mL. Splenocytes (0.1/well) were cultured intriplicate on 96-well flat-bottom culture plates. Splenocytes (0.1mL/well) were stimulated with ConA (1×10⁻² g/L, 0.1 mL/well) to induceIL-2, IL-4 and IFN-γ production. In other plates, splenocytes werecultured with lipopolysaccharide (1×10⁻⁶ g/L) to induce IL-6 production.These plates were incubated at 37° C., 5% CO₂; the culture time forIL-2, IL-4 and IL-6 is 24 hours (or 72 hours for IFN-γ). Afterincubation, supernatants were collected and stored at −70° C. Thecytokines were determined by sandwich ELISA as previously describedusing a commercial kit.

Mitogenesis of Splenocytes

Splenic T and B-cell proliferation was determined by 3H-thymidineincorporation according to the method previously described (Chouaib, S.;Welte, K.; Mertelsman, R.; Dupont, B.; J. Immunol., 1985, 135,1172-1179). 0.1 mL splenocytes in CM (2×10⁶ cells/mL) were cultured in96-well flat-bottom cultured plates with ConA or LPS (10 μg/mL) orwithout ConA or LPS (Spontaneous mitogenesis). They were incubated at37° C., 5% CO₂ for 48 hours and then pulsed with 3H-thymidine(0.5/well). After 24 hours, supernatants were harvested by a cell sampleharvester.

Vitamin E Measurement

Vitamin E levels in mouse liver were measured by HPLC according to themethod described previously (Lee, J., Jiang, S., Liang, B., Inserra, P.,Zhang, Z., Solkoff, D., Watson, R. R, Nutr. Res., 1998, 18, 327-339).0.2 g of tissue was homogenised in 1 mL of water. Butylatedhydroxytoluene was added to prevent oxidation of α-tocopherol.α-Tocopherol was extracted from the homogenate using pentanes, ethanoland sodium dodecyl sulfate and concentrated under a steady flow ofnitrogen gas at 20° C. The concentrate was redissolved in 0.5 mL ofmethanol and injected onto a C18 column (3.9×150 mm). A mobile phasecomposed of methanol: 1 mol/L sodium acetate in the ratio of 97:3 (byvolume) at a flow rate of 1.5 mL/min was used. α-Tocopherol, eluted at22 min and was monitored by a fluorescence detector (Millipore) at290-nm excitation and 320-nm emission wavelength.

The results indicated that orally ingested feijoa fruit extract at adose of 1.8 mg/day per aged mouse, decreases splenic T-cell productionof IL-4, TNF-α and TNF-β. IL-2 and IFN-γ production was notsignificantly affected in either the control group or the group giventhe supplement at a dosage of 1.8 mg/day. Splenocyte proliferation whenstimulated by ConA at a concentration of 2 μg/mL also decreased. Thefeijoa fruit extract supplementation at an appropriate dose causedsignificant increases in splenic T-cell immune function in aged mice(Table 3).

TABLE 3 Young Aged Aged Unsupplemented Unsupplemented supplementedT-cell proliferation (%) 130 ± 15* 100 ± 9  78 ± 9* (aged unsupplemented= 100) B-cell proliferation (%) 130 ± 8*  100 ± 4  85 ± 4* (agedunsupplemented = 100) IL-2 level by splenocytes 278 ± 26  264 ± 38 216 ±47  (pg/mL) IFN-γ level by splenocytes 1555 ± 12  750 ± 37 907 ± 12 (pg/mL) TNF-α level by splenocytes 290 ± 30* 650 ± 53 450 ± 35* (pg/mL)TNF-β level by splenocytes  95 ± 20* 142 ± 18  70 ± 10* (pg/mL) IL-4level by splenocytes 135 ± 40  165 ± 45  90 ± 30* (pg/mL) MDA levels inliver tissue  0.12 ± 0.02* 0.325 ± 0.07  0.19 ± 0.03* (mol/mg protein)Hepatic vitamin E level (%) 200 ± 26* 100 ± 9  177 ± 3*  (agedunsupplemented = 100) Data indicates mean ± SD from 6 mice per group.*Shows the statistical significance compared to aged control determinedby unpaired Students t-test.

Aged mice supplemented with feijoa fruit extract at a dosage of 1.8mg/day showed a decrease in their IL-4 production by 44% (P<0.01) versusthe aged unsupplemented group.

Aged mice supplemented with feijoa fruit extract showed a 20% decreasein ConA stimulated splenic T-cell mitogenesis vs. aged mice not fed thefeijoa fruit extract. Similarly, mice supplemented with feijoa fruitextract showed a 14% decrease in LPS stimulated splenic B-cellmitogenesis. Aged animals often have spontaneously stimulated B-cells,which do not function as well as those in younger animals and alsoinhibit T-cells. Therefore, lowering mitogenesis or cell division byB-cells should be beneficial to host defenses.

Immunosenescence (deterioration of the immune system by age advancement)is a major contributing factor in survival to old age or premature deathin humans and animals. Some of the adverse effects include dysregulatedcell division of T- and B-lymphocytes upon stimulation by mitogens invitro, or pathogens in vivo with altered cytokine production. In thepresent studies, B- and T-lymphocytes from aged mice divided less thanthose of young mice, and those from aged mice fed the feijoa fruitextract. The key observations on regulatory cytokines includestimulation of INF-γ, TNF-α, -β and -γ, and IL-4 by consequences ofimmunosenescence in aging. The lowering of these cytokines due toconsumption of dietary feijoa fruit extract suggests better overallimmune regulation, which can provide improved disease resistance.

Immune regulation during immunosenescent T-cell immune dysfunction seemsto be associated in part with the level of vitamin E. Vitamin E reversesage-associated defects in T-cells, particularly naïve T-cells whoselevels are greatly reduced in aging. Vitamin E directly affects T-cellsas well as PGE2 inhibition. Without wishing to be bound by theory, theapplicant hypothesises that the beneficial effects of feijoa fruitextract supplementation that are observed on immunosenescence that causeT-cell immune dysfunction could be due, in part to its ability toconserve cellular vitamin E level. Aged mice supplemented with feijoafruit extract showed a 77% increase in the level of hepatic vitamin Ewhen compared to the aged unsupplemented test group (Table 3). Feijoafruit extract also lowered TNF-α which is known to be elevated in aging.Thus feijoa fruit extract has beneficial influence in moderating immunefunctions in the aged.

Example 4: Human Clinical Trial to Measure Hypertension,Hyperlipidaemia, and Hyperglycaemia

The study population consisted of men and women, 40-75 years of age,with non-insulin dependent Type-2 diabetes. Exclusion criteria includedType-1 diabetes; use of any supplements other than single dailymultivitamin; having any major illness such as cancer, asthma, or heartfailure; any previous cardiac events; pregnancy, or being a nursingmother. The protocol of this 12-week, randomised, double-blind,placebo-controlled trial was approved by the institutional review boardat Mashad University and performed in accordance with the Declaration ofHelsinki. Study subjects were recruited by the university diabetesclinic. All subjects gave written informed consent before participationin this research trial. Subjects were randomly assigned to receiveeither feijoa fruit extract (150 mg, once a day) in the form of a pillor a matched placebo, both of which remain constant throughout the studyperiod. The feijoa extract was prepared according to the process set outin Example 1.

TABLE 4 Feijoa fruit extract: Human participant study. Placebo Feijoafruit extract Number of participants 14 20 Male:female ratio 7:7 4:16Age (years) 52.6 55.0 Weight (kg) 77.6 77.2 Height (cm) 164.4 158.6Participants received 150 mg/person/day of the feijoa fruit extract. Asshown in Table 4 above, the demographic and clinical characteristics aresimilar. Mean years of diabetes treatment was the same for both groups.19 of the 20 subjects supplemented with feijoa extract used additionalmedication: 95% used a combination of Metformin and Glibenclamide withtwo participants using insulin. 13 of the 14 subjects in the placebogroup used additional medication: 55% used Metformin and Glibenclamide,insulin (1 subject) or Atenolol.

Blood pressure and heart rate were recorded at baseline and after 12weeks. Blood pressure and heart rate were measured on the left arm after10 min rest. Korotkoff sounds I and V were taken as the systolic anddiastolic blood pressures, respectively. Repeated readings were taken at2-minute intervals for a total of 3 sitting measurements and the averagewas recorded. All treatments including anti-diabetic medicationsremained constant throughout the study period. Unused pills werecollected and counted at monthly follow-up visits to assess participantcompliance. Changes in concomitant medications and clinical adverseeffects at follow up visits were investigated by questioning theparticipants, none were reported.

Analysis was performed according to the intention to treat principle.Thus all randomised patients who received at least one dose of studytreatment and who have both a baseline and at least one post-baselinemeasurement were analysed. The data are expressed as mean±SEM.Statistical analyses are performed with SPSS version 11.5 (SPSSInstitute, Chicago, Ill.) (Sprinthall R. C., Basic statistical analysis,Boston: Allyn and Bacon; 2003, 550-565). Chi-square test with Yates'correction is used for non-continuous variables for the prevalencestudy. Student's t-test is used to assess the statistical significanceof the continuous variables. Comparable nonparametric test (Mann-WhitneyU test) is substituted when tests for normality and equal variancefailed. A value of P<0.05 was used as a criterion for statisticalsignificance. Trial participants showed no significant changes in thestandard liver enzyme profile due to the feijoa fruit extract. Inaddition, there is no significant difference in serum creatinine,albumin, and urea (data not shown) due to the feijoa fruit extract.These data suggest no toxicity occurs.

Initial systolic blood pressure was comparable for the placebo group andthe group supplemented with feijoa fruit extract. There were nosignificant differences observed in HDL cholesterol and diastolic bloodpressure. The intake of the feijoa fruit extract induced a decrease insystolic blood pressure in comparison with the placebo group whichshowed an increase (P<0.0776). This indicated an improvement of systolicblood pressure control with feijoa fruit extract at the end of the 12week trial period when compared to placebo (Table 5). Comparatively,diastolic blood pressure levels trended lower in the group receiving thefeijoa fruit extract while the placebo group increased (P<0.35).

TABLE 5 The effect of feijoa fruit extract in human subjects with Type-2diabetes. Placebo Feijoa fruit extract Variable Baseline Week 12 ChangeBaseline Week 12 Change Triglycerides  208.2 ± 23.5 241.9 ± 29.7 +33.6 ±12.6 197.9 ± 28.1 159.8 ± 20.8* −38.1 ± 20.9 (mg/dL) Glucose (mg/dL)168.4 ± 8.4 181.9 ± 10.3 +13.5 ± 30.7 184.0 ± 8.2  147.7 ± 8.0*† −36.3 ±2.7  HbA1c (%)  7.5 ± 0.4  8.1 ± 0.4 +0.49 ± 0.19  8.6 ± 0.3  7.7 ± 0.2*−0.86 ± 0.14 LDL-cholesterol  109.3 ± 37.2 116.1 ± 40.3 +6.8 ± 7.5 105.2± 15.2 87.2 ± 8.7  −18.1 ± 14.4 (mg/dL) Serum cholesterol 194.8 ± 9.6209.8 ± 10.1 15.0 ± 9.8 201.1 ± 9.9  183.6 ± 7.3  −17.5 ± 6.9  (mg/L)Systolic BP 144.2 ± 0.2 142.9 ± 0.5  +0.36 ± 1.5  144.3 ± 1   137.6 ±1.3  -6.7 ± 2.7 (mmHg) Analysed by Student's t-test, *P <.001 comparedwith the baseline, †P <.001 compared with the placebo group.Measurements were performed on serum samples after 8-hours of fasting atthe baseline and after 12 weeks of treatment. Values are means ± SEM (n= 20 subjects in feijoa fruit extract group and 14 in placebo except forLDL-cholesterol when both groups are 6).

Supplementation with feijoa fruit extract also reduced the values ofseveral serum variables which were used to assess the health status ofdiabetics. Subjects receiving the feijoa fruit extract showed areduction in fasting serum glucose whereas the placebo group showed anincrease (P<0.0001) (Table 5). Significant changes to HbA1c levels werealso observed by the 12^(th) week of the trial. Mean HbA1c levels in thefeijoa fruit extract treated group decreased while the levels in theplacebo group increased (P<0.0001) (Table 5). In addition, at the end ofthe 12-week trial period, patients supplemented with feijoa fruitextract show a marked decrease in serum triglycerides (P<0.0127), serumtotal cholesterol (P<0.0887) and LDL-cholesterol (P<0.157) (Table 5). Insummary, these data show that feijoa fruit extract can improve diabetescontrol, reduce antihypertensive medicine use, and may favour areduction in cardiovascular disease risk in individuals with Type-2diabetes.

Example 5: Obesity Study Using Obese Mice

Male leptin-deficient (ob/ob) obese mice (5 weeks old, 26˜33 g) and leanC57BL/6J wild type (WT) mice were purchased from SLC, Inc. (Hamamtsu,Japan). The mice were housed in temperature (22±2° C.), humidity (50±5%)and light (12 hr light/dark cycles) controlled conditions. The animalswere allowed to acclimatize for 1 week prior to experiments. Then themice were randomly divided into three groups of 6 mice. Negative control(genetically normal control, C57BL/6J) and positive control (ob/obcontrol) groups were fed normal American Institute of Nutrition (AIN)93M diet, and the other group was fed AIN93M diet containing feijoaextract at 300 mg/kg diet for 16 weeks. The feijoa fruit extractprepared in accordance with Example 1 was mixed homogenously into thepowdered diet and the diet was given in the solid form. Body weight ismeasured at the beginning of the experiment and weekly intervals for the16 week study period. The amount of food consumed by each group wasrecorded on a daily basis.

After 16 weeks on experimental diets, the mice were sacrificed andtissues are collected for analysis. Blood was collected, dispensed intoserum tubes and centrifuged for 20 min at 1,600 rpm. The serumtriglyceride and total cholesterols levels were analysed by commercialkits (Cayman Chemical, Ann Arbor, Mich., USA) and high-densitylipoprotein-cholesterol were measured fluorimetrically with quantitationkit (BioVision Research Products, Mountain View, Calif., USA).

The changes in body weight during the experimental period due to theeffect of feijoa fruit extract are shown in Table 6. Feijoa fruitextract supplementation significantly decreased the amount of weightgained by 24% when compared to the control in ob/ob mice. Throughout theexperiment, the food intake was higher for the obese control group thanin the control group. However, the obese mice in both the control groupand feijoa fruit extract supplemented group consumed similar volumes offood. The food efficiency ratio significantly decreased in a feijoafruit extract supplementation group compared to obese control group.

TABLE 6 Body Weight Changes in Obese Mice. Feijoa fruit Normal Ob/obcontrol extract treated mice control (obese) ob/ob mice Initial bodyweight (g) 21.79 ± 1.09*  32.45 ± 2.15  34.40 ± 1.96  Final body weight(g) 37.29 ± 3.11*  65.23 ± 3.97  59.05 ± 2.72* Weight gain (g/day) 0.14± 0.03* 0.29 ± 0.05  0.22 ± 0.04* Food Intake (g/day) 3.53 ± 0.35* 5.28± 0.39 4.89 ± 0.58 Food efficiency ratio 3.90 ± 0.49* 5.51 ± 0.57  4.49± 0.37* (body weight gain/ food intake) × 100 Heart weight (g) 0.13 ±0.01  0.13 ± 0.01 0.13 ± 0.01 Kidney weight (g) 0.22 ± 0.02* 0.27 ± 0.020.25 ± 0.02 Liver weight (g) 1.89 ± 0.17* 4.44 ± 0.39  3.97 ± 0.31*Spleen weight (g) 0.07 ± 0.01  0.07 ± 0.01 0.06 ± 0.01 Retroperitoneal0.53 ± 0.19* 3.15 ± 0.46 2.63 ± 0.36 adipose tissue weight (g)Epididymal adipose 1.58 ± 0.26* 4.23 ± 0.36  3.63 ± 0.43* tissue weight(g) Values are means ± SD from 6 mice/group. Mean with * indicates asignificant difference at p < 0.05, compared to negative control.

In the feijoa fruit supplemented group, the weights of the heart,kidney, and spleen were not significantly different when compared tocontrol group (Table 6). In mice supplemented with feijoa fruit extract,the weight of the liver was 10.6% less than that of the control group.In order to examine the effect of feijoa supplementation on body fataccumulation, the weight of adipose tissue in ob/ob mice was measured.The weights of retroperitoneal and epididymal adipose tissues weresignificantly higher in the ob/ob control group when compared to normalcontrol mice. The weight of epididymal adipose tissue in the feijoafruit extract group was significantly lower than the ob/ob controlgroup. However, the supplementation of feijoa fruit extract did notappear to have an effect on retroperitoneal adipose tissue in the feijoafruit extract supplemented group when compared to the obese mice.

It was also found that the ob/ob control group had significantlyelevated serum total cholesterol and triglyceride levels when comparedto the normal control group. However, it was found that supplementationwith feijoa fruit extract did not affect the serum total cholesterol andtriglyceride levels. In addition, HDL-cholesterol levels were notchanged by supplementation of feijoa fruit extract compared to ob/obcontrol group. Supplementation in the diet of obese mice with the feijoafruit extract was found to reduce the hepatic total cholesterol levelcompared to the ob/ob control mice; however the hepatic triglyceridelevel was not affected.

Obese mice fed a diet supplemented with feijoa fruit extract did notgain as much body weight, showed a lower food efficiency ratio, andexhibited significant beneficial changes in hepatic total cholesterollevels. This tendency is presumed to relate to the suppression of lipidaccumulation in adipose tissues. Thus, dietary supplementation of feijoafruit extract may physically affect body weight gain and reduce fattissue accumulation.

Example 6: Arthritis Studies Using Collagen-Induced Arthritis Mice

Male DBA/1J mice (6 weeks old, 20˜22 g) were purchased from Japan SLC,Inc. (Shizoka, Japan). The mice were divided into 4 groups of 5 mice pergroup and were fed American Institute of Nutrition 93G diet and water adlibitum at a temperature of 25° C. at relative humidity of 55%. Micewere maintained in a 12 hour light/dark cycle under specificpathogen-free conditions according to the institutional instructions ofKyung Hee University, Korea. All animal experiments were performed inaccordance with ethical guidelines issued by the Animal Care and Usecommittee of the College of Medicine, Seoul National University (Seoul,Korea). The 4 groups were normal control (no collagen-induced arthritis,no dietary supplementation), negative control (collagen-inducedarthritis receiving no dietary supplement), positive control(collagen-induced arthritis diet supplemented with 10 mg/kgmethotrexate) and feijoa fruit extract supplemented (collagen-inducedarthritis, diet supplemented with feijoa fruit extract powder). The bodyweights between the mice groups did not show any significantdifferences. There were no significant differences between the volumesof food and water that are consumed between the groups. This indicatesthat changes in experimental data were not due to either body weight orwater and food consumption. An average 25 g mouse consumed approximately6 g of diet per day, consuming 1.8 mg of feijoa fruit extract. Arthritiswas induced by injecting 0.1 ml of bovine Type II collagen (Chondrex,Inc., Redmond, Wash., USA) was dissolved in 10 mM acetic acid atconcentration of 2 mg/mL by stirring overnight at 4° C. The collagen wasemulsified in an equal volume of complete Freund's adjuvant containingMycobacterium tuberculosis in an ice-cold water bath. Mice are injectedsubcutaneously at the base of the tail with 0.1 mL of emulsion. The micewere given a booster collagen injection on day 21 with incompleteFreunds adjuvant of 0.1 ml of emulsion. Clinical assessment ofinflammation on the paws of the mice was visually performed 3 timesweekly between day 56 and day 79 using a 4-point scale (0-4) for eachpaw (Table 1). The total score for clinical assessment was measured oneach mouse (4 points per paw with maximum score of 16). A mouse modulartreadmill with 10 inches of running surface made by Columbus Instruments(USA) was used. The speed is adjusted from 6-100 meters per minute toassess the mouse's maximum speed ability. Running speed and footpressure measurements were performed 3 times weekly from day 56 untilday 79. The mice were sacrificed at the end of the trial. The knees ofthe mice were removed, frozen, and later sectioned. They are stainedwith hematoxylin and eosin and photographed as shown in FIG. 2. The hindpaws are structurally assessed with X-ray micro computerised tomographicimaging within inflamed joints and are photographed with intensity shownin colour. They were perfused with radiopaque silicone rubber, theuptake of silicon is measured by X-ray micro CT at 21 μm resolution andimages were analyzed in 3-D using custom software and laboratorymicro-CT scanner consisting of a tungsten-anode X-ray tube with arelatively small focal spot (˜10 μm), coupled to a high-resolution X-raydetector system (˜50 μm pixel spacing). Uptake of the radiopaquesilicone rubber reduces X-ray penetration and changes colour intensity.

The progression of arthritis was assessed by subjecting mice to atreadmill test and comparing the levels of inflammation between allgroups (Table 7 and Table 8). Slower running speeds and lighter footpressures are indicative of inflammation and an attempt by the mouse toreduce any pain. As shown in Table 7, subjects treated with feijoa fruitextract showed beneficial clinical scores when compared to the arthriticcontrol and a similar value when compared to the methotrexate treatedgroup. As expected, running speed was highest in normal control group,and lowest in the arthritis-induced control group (Table 8).Surprisingly and advantageously there was no significant differencebetween the methotrexate and the feijoa fruit extract treated group.

TABLE 7 Assessment of feijoa fruit extract on paw swelling in arthritisinduced mice Arthritic Methotrexate Feijoa fruit Normal control controltreated extract Clinical Score 11.2 ± 0.7* 5.5 ± 1.2 10.5 ± 0.7* 9.5 ±2.0* *indicates a statistical significance determined by student'st-test compared to arthritic control

TABLE 8 Effects of Feijoa fruit extract on running speed and footpressure Running Foot Treadmill Results Speed (mm/s) pressure Normalcontrol 133.05 ± 3.72* 148.54 ± 2.39* Arthritic control 91.24 ± 2.59136.89 ± 0.85  Methotrexate treated 121.36 ± 1.69* 141.23 ± 5.28* Feijoafruit extract 120.54 ± 5.42* 143.97 ± 3.69* Values are means ± SD from 5mice/group. *indicates a significant difference at p < 0.05, compared tonegative control.

The mice were sacrificed at the end of the trial period and the jointsexamined for the progression of arthritis. Histological evaluation ofthe joint sections of CIA mice showed decreased synovial hyperplasia (anincrease in destructive cytokine production in synovial fluid) (FIG. 3).In contrast, the feijoa fruit extract and methotrexate treated groupshad relatively normal joint structures with little soft tissue swelling(FIGS. 2, 3), while normal control showed no synovial thickening orinflammatory cell infiltration, and the joint structures were wellmaintained. Since synovial hyperplasia results in bone and cartilageerosion which can facilitate the onset of rheumatoid arthritis, thereduction observed in the feijoa extract supplemented mice compared tothe arthritic control suggests that the extract is beneficial inreducing the effects and onset of rheumatoid arthritis.

Radiologic studies revealed that there was no evidence of jointdeformity or soft tissue swelling in the normal control. Severe jointdestruction along with soft tissue swelling was observed in thearthritic control group; however, members of the feijoa treated grouphave relatively normal joint structures with little soft tissue swellingwhich was comparable with the methotrexate group of mice.

To establish whether the protective effect of the feijoa fruit extractwas related to the anti-inflammatory response observed in the CIA mice,pro-inflammatory cytokines (TNF-α, IL-2 and INF-γ) were investigatedusing the ELISA assay described in Example 3 (Table 9). It is well knownthat such cytokines play crucial roles in joint destruction by damagingthe synovial cells within the joint. Consequently, a reduction insynovial cells within the joint results in an increase in friction,which in turn, results in inflammation and the potential development ofarthritis. As shown in Table 9, the feijoa fruit extract treated grouphad significantly lower levels of pro-inflammatory cytokines whencompared to the arthritic control and showed comparable values whencompared to mice treated with methotrexate. Supplementation with feijoafruit extract resulted in significant decreases in IL-2 and IFN-γ levelsby 48% and 40% respectively when compared to the arthritic controlgroup, indicating reduced destruction within the joint.

TABLE 9 Cytokine data for CIA mice. Normal Arthritic Methotrexate Feijoafruit control control treated extract TNF-α level by 200 ± 22  536 ± 60255 ± 15* 233 ± 46* splenocytes (pg/mL) IL-2 level by 50 ± 16 151 ± 12104 ± 10  90 ± 8* splenocytes (pg/mL) IFN-γ level by 76 ± 12 394 ± 76182 ± 21* 200 ± 14* splenocytes (pg/mL) Values are means ± SD from 5mice/group. Mean with * indicates a significant difference at p < 0.05,compared to arthritic control.

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention.

Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically referred to in thespecification.

What we claim is:
 1. A method of: preventing or treating hyperglycaemiain a patient; or preventing or ameliorating one or more symptoms ofdiabetes associated with hyperglycaemia in a patient; comprisingadministering to the patient an effective amount of feijoa fruitextract, thereby: preventing or treating hyperglycaemia in the patient,or preventing or ameliorating the one or more symptoms of diabetesassociated with hyperglycaemia in the patient.
 2. The method of claim 1,wherein the patient's serum glucose is lowered.
 3. The method of claim2, wherein HbA1c levels in the patient are reduced.
 4. The method ofclaim 2, wherein the patient is a human patient with Type-2 diabetes. 5.The method of claim 1, wherein the feijoa fruit extract comprises one,two, or more compounds selected from the group consisting of: catechin,epicatechin, gallocatechin, proanthocyanidins, flavone, ellagic acid,pedunculagin, and other feijoa fruit ellagitannins.
 6. The method ofclaim 1, wherein the feijoa fruit extract comprises flavones and/orpedunculagin.
 7. The method of claim 1, wherein the feijoa fruit extractcomprises a whole fruit extract.
 8. The method of claim 1, wherein thefeijoa fruit extract is prepared by extraction with water or byextraction with a combination of water and ethanol.
 9. The method ofclaim 1, wherein the feijoa fruit extract is prepared by a processincluding the steps of: (i) contacting feijoa fruit, or feijoa fruitskin, or feijoa fruit pulp or residues of feijoa fruit from feijoa fruitjuicing, with water or a combination of water and ethanol to provide anaqueous extract, or aqueous/hydrophilic extract, and a solid residue;(ii) separating the aqueous extract, or aqueous/hydrophilic extract,from the solid residue to give a crude aqueous feijoa fruit extract, ora crude aqueous/hydrophilic feijoa fruit extract; (iii) evaporating thecrude aqueous feijoa fruit extract, or crude aqueous/hydrophilic feijoafruit extract from step (ii) to give a substantially aqueous feijoafruit extract concentrate and a precipitate; (iv) separating thesubstantially aqueous feijoa fruit extract concentrate from theprecipitate; and (v) fractionating the substantially aqueous feijoafruit extract concentrate from step (iv) to concentrate at least onecomponent thereof.
 10. The method of claim 9, wherein the feijoa fruitextract comprises flavone and/or pedunculagin.
 11. The method of claim9, wherein the feijoa fruit extract comprises at least about 70 weightpercent polyphenol compounds, or at least about 80 weight percentpolyphenol compounds.
 12. The method of claim 9, wherein the feijoafruit extract is formulated as a nutraceutical or pharmaceuticalcomposition comprising the feijoa fruit extract and one or morenutraceutical or pharmaceutically acceptable excipients.
 13. The methodof claim 9, wherein the feijoa fruit extract is formulated as a dosageunit comprising: (i) about 50 mg to about 500 mg feijoa fruit extract,or (ii) about 150 mg feijoa fruit extract, wherein the feijoa fruitextract is formulated as a powder.
 14. A method of treating orpreventing Type-2 diabetes in a patient comprising administering to thepatient the feijoa fruit extract prepared by extraction with water or byextraction with a combination of water and ethanol, wherein the feijoafruit extract comprises a whole fruit extract, thereby treating orpreventing the Type-2 diabetes in the patient.
 15. The method of claim14, wherein the feijoa fruit extract comprises flavone and/orpedunculagin.
 16. The method of claim 14, wherein the feijoa fruitextract comprises at least about 70 weight percent polyphenol compounds,or at least about 80 weight percent polyphenol compounds.
 17. The methodof claim 14, wherein the feijoa fruit extract is formulated as a dosageunit comprising: (i) about 50 mg to about 500 mg feijoa fruit extract,or (ii) about 150 mg feijoa fruit extract, wherein the feijoa fruitextract is formulated as a powder.